Tubular fluid flow in the kidney causes drag on the apical surface of epithelial cells and these forces modulate cytoskeletal stress. This in turn activates multiple intracellular signaling pathways causing reorganization of cytoskeleton and cell remodeling. Our experiments allowed us to dissect the reorganization into several distinct stages (). The process seems to involve a Ca2+ influx through MSCs.
The initial decrease in cytoskeletal stresses is consistent with our previous findings that sudden change in shear stress causes reversible compression in α-actinin. Repetitive application results in progressive adaptation 
. The shear stress amplitude in this study is an order of magnitude smaller than in our previous study indicating that we are far from saturation in these experiments. F-actin shows no significant change within this time suggesting that this process only involves the conformational changes of the linking proteins or deformation of actinin itself (). This adaptation to mechanical stress has also been observed with magnetic beads attached to the cell surface 
The shear-induced increase in cytoskeletal stress and corresponding inward contraction of F-actin with time appears to be an essential process for reorganization of the cytoskeleton since this is followed by extended reduction in cytoskeletal stresses and redistribution of F-actin to the periphery. Similar increase in cytoskeletal stress (as in Phase-II) has also been observed using magnetic beads at early stages of cell adaptation 
. Recently, Martin, et. al.
reported that pulsed apical contractions powered by actomyosin pull the cytoplasm inward towards the nucleus 
. This contractile force is known to facilitate actin polymerization via actin binding proteins at focal adhesions 
. Thus, the observed inward movement of actin during phase II () indicates the contraction of actin network that triggers the subsequent actin reassembly. Our results thus suggest that such actin-myosin contraction can be activated by flow shear stress so that the net effect of fluid shear stress acts via body stress in the cell.
Long term adaptation seems to involve Rho GTPases and its downstream targets. Rho-associated kinase (ROCK) is a key regulator for cytoskeleton reorganization, focal adhesion development and cell motility 
. In MDCK cells, application of 20 µM Y27632, a Rho-ROCK inhibitor, caused disassociation of F-actin, and blocked the cytoskeleton remodeling in flow (Fig. S2
). Our results suggest Rho GTPases may participate in flow induced actin reorganization. It has been reported that applying a mechanical force to the cell surface of MDCK cells leads to Rho translocation 
, and Rho-kinase regulates myosin light chain phosphorylation that increases cell contractility and alters the formation of actin stress fibers 
. The observed contractile force in phase-II () seems to be the result of Rho mediated myosin contraction that facilitates the actin restructuring. In addition, flow-induced Rho activation facilitates cytoskeleton reorganization in endothelial cells 
with an initial transient inactivation that is followed by an increase peaking at ~60 minutes 
. Although this time scale (in the range of 5–60 min) is similar to our observation for actin disassociation and reassembly under flow (), the cytoskeletal dynamics are different for endothelial and epithelial cells probably because they have different cytoskeletal prestress and organization 
The whole adaptation process can be initiated by MSCs since changes in structure and motility were blocked by Gd3+
and GsMTx4. A similar effect has been reported with Gd3+
for stresses from magnetic beads 
. Flow-induced MSC activation and Ca2+
transients have been observed in MDCK cells 
. We have also shown that shear stress causes a transient Ca2+
response in MDCK cells and the Ca2+
influx can be blocked by MSC inhibitors Gd3+
and GsMTx4 (Fig. S1
). The time dependence of the Ca2+
response shows that intracellular Ca2+
peaked within 1 min in flow, preceding the contractile actin ring formation. These observations suggest a connection between Ca2+
increase and downstream signaling pathways, such as Rho GTPases activation. The reorganization, however, does not require a continued elevation of Ca2+
, but the transient elevation appears to lead to activation of a long lived later messenger such as Rho or possibly altered prestress in the cytoskeleton.
In conclusion, fluid shear stress regulates cytoskeletal dynamics triggered by Ca2+ permeable MSCs and progressing through a series of structural adaptations, leading to a reduction of cytoskeletal tension. The process is illustrated schematically in . This remodeling is important for the cell to minimize the stress that it experiences and/or to optimize its structure to resist the external force so as to minimize the internal stress gradients.
Schematic of proposed multiphase-adaptation to fluid shear stress in epithelial cells.